Anaerobic mineralization of toluene by enriched soil-free consortia with solid-phase humin as a terminal electron acceptor

The anaerobic biodegradation of toluene proceeds very slowly owing to limited electron acceptors in contaminated aquifer. The liquid reagents traditionally used to enhance this process readily migrate away from the contaminated site, and continuous addition would cause secondary pollution. In our previous study, the reduced solid-phase humic substances (humin), which are redox active, were found to act as electron donors to promote the microbial reactions. Here, we provide new evidence that humin can promote the anaerobic biodegradation of toluene as a terminal electron acceptor. When inoculating nitrate-reducing (NR) and iron-reducing (IR) consortia with toluene degradation activities, the average toluene degradation rates reached 21.20 ± 1.18 μmol/(L·d) and 15.43 ± 0.41 μmol/(L·d) in the presence of a sediment humin (HMcj), and 94.69% ± 4.26% and 93.20% ± 3.73% of the electrons released from toluene oxidation to CO2 could be recovered by the reduction of HMcj, respectively. Spectroscopy analyses revealed that quinone moieties and nitrogen-containing moieties may be the electron-accepting groups of HMcj. Based on 16S rRNA sequencing, Cellulomonas spp. were the possible functional bacteria in the culture with NR consortium as the inoculum, while Azospira spp., Cellulomonas spp. and Bacillus spp. were the possible functional bacteria in the culture with IR consortium as the inoculum. Further Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analyses indicated that toluene oxidation and extracellular electron transfer functions were more abundant in HMcj amended cultures, suggesting a possible enhancement mechanism by HMcj. Additionally, experiments using natural groundwater illustrated that toluene degradation was highly dependent on its concentration, HMcj dosage, pH, and salinity. The study of a column filled with HMcj-coated quartz sand demonstrated a desirable level of toluene degradation in a continuous-flow mode without the presence of other electron acceptors. This study provided an effective and green approach for the remediation of the toluene-contaminated groundwater.